The present invention relates to gene promoter sequences isolated from bell pepper and their use to regulate chimeric gene expression in plants. In particular it describes the isolation and use of DNA sequences which permit a high level of expression of foreign genes in transgenic plants.
The expression of genes in plants is controlled by a number of regulatory components, including nucleic acid and protein elements. Where the plant gene exists as double stranded DNA, the primary steps of expression involve the production of a messenger RNA by a polymerase enzyme. The initiation of this part of the expression process is controlled by a region commonly referred to as the xe2x80x9cpromoterxe2x80x9d. The promoter lies upstream (5xe2x80x2) of the protein encoding region and may be constitutive or tissue-specific, developmentally-regulated and/or inducible.
Within the promoter region there are several domains which are necessary for full function of the promoter. The first of these domains lies immediately upstream of the structural gene and forms the xe2x80x9ccore promoter regionxe2x80x9d containing consensus sequences, normally 70 base pairs immediately upstream of the gene. The core promoter region contains the characteristic CAAT and TATA boxes plus surrounding sequences, and represents a transcription initiation sequence which defines the transcription start point for the structural gene. The precise length of the core promoter region is indefinite but it is usually well-recognisable. Such a region is normally present, with some variation, in all promoters. The base sequences lying between the various well-characterised xe2x80x9cboxesxe2x80x9d appear to be of lesser importance.
The presence of the core promoter region defines a sequence as being a promoter: if the region is absent, the promoter is non-functional. Furthermore. the core promoter region is insufficient to provide full promoter activity. A series of regulatory sequences, usually upstream of the core, constitute the remainder of the promoter. The regulatory sequences determine expression level, the spatial and temporal pattern of expression and, for an important subset of promoters, expression under inductive conditions (regulation by external factors such as light, temperature, chemicals, hormones). Manipulation of crop plants to alter and/or improve phenotypic characteristics (such as productivity or quality) requires the expression of heterologous genes in plant tissues. Such genetic manipulation therefore relies on the availability of means to drive and to control gene expression as required; for example, on the availability and use of suitable promoters which are effective in plants and which regulate gene expression so as to give the desired effect(s) in the transgenic plant. It is advantageous to have the choice of a variety of different promoters so that the most suitable promoter may be selected for a particular gene, construct, lo cell, tissue, plant or environment.
Promoters (and other regulatory components) from bacteria, viruses, fungi and plants have been used to control gene expression in plant cells. Numerous plant transformation experiments using DNA constructs comprising various promoter sequences fused to various foreign genes (for example, bacterial marker genes) have led to the identification of useful promoter sequences. It has been demonstrated that sequences up to 500-1000 bases in most instances are sufficient to allow for the regulated expression of foreign genes. However, it has also been shown that sequences much longer than 1 kb may have useful features which permit high levels of gene expression in transgenic plants. A range of naturally-occurring promoters are known to be operative in plants and have been used to drive the expression of heterologous (both foreign and endogenous) genes in plants: for example, the constitutive 35S cauliflower mosaic virus promoter, the ripening-enhanced tomato polygalacturonase promoter (Bird et al, 1988, Plant Molecular Biology, 11:651-662), the E8 promoter (Diekman and Fischer, 1988, EMBO, 7:3315-3320) and the fruit specific 2A11 promoter (Pear et al, 1989, Plant Molecular Biology, 13:639-651) and many others.
As stated above, successful genetic manipulation relies on the availability of means to control plant gene expression as required. The scientist uses a suitable expression cassette (incorporating one or more promoters and other components) to regulate gene expression in the desired manner (for example, by enhancing or reducing expression in certain tissues or at certain developmental stages). The ability to choose a suitable promoter from a range of promoters having differing activity profiles is thus important.
In the present invention, we have isolated and fully sequenced the fibrillin gene promoter and the capsanthin-capsorubin synthase gene promoter from bell pepper (Capsicum annuum). The fibrillin promoter (FIB) essentially controls the production of the protein known as xe2x80x9cfibrillinxe2x80x9d in peppers. This fibrillin protein is associated with the plant chromoplasts and is involved in the packaging and organisation of carotenoids. It is the FIB promoter that regulates the accumulation of fibrillin during chromoplast differentiation.
The capsanthin-capsorubin synthase promoter (CCS) controls the production of the enzyme capsanthin-capsorubin synthase. This enzyme catalyses the conversion of the ubiquitous 5,6-epoxycarotenoids, antheraxanthin and violaxanthin, into capsanthin and capsorubin, respectively. It is the CCS promoter that specifically regulates the CCS gene during chloroplast to chromoplast differentiation.
The present invention aims to provide, inter alia, alternative promoters capable of driving gene expression in plants. We believe the present invention provides new developmentally regulated promoters which may be particularly useful in controlling chimeric gene expression in particular parts of a plant at a specific stage during development e.g. fruit ripening. This may be especially useful in plants such as tomato plants.
According to the present invention, there is provided a DNA sequence encoding a bell pepper fibrillin gene promoter capable of driving gene expression in plants having the sequence shown in SEQ ID NO 1 or active variants thereof.
Further according to the present invention, there is provided a DNA sequence encoding a bell pepper capsanthin-capsorubin gene promoter capable of driving gene expression in plants having the sequence shown in SEQ ID NO 2 or active variants thereof.
The cDNA sequence of the bell pepper FIB gene from the ATG initiation codon to a position 214 bp upstream, and of the CCS gene from the ATG initiation codon to a position 200 bp upstream have been previously described by Deruere et al (1994) (Biochem. Biophys. Res.Commnun. 199 (3) 1144-50). Similarly, the cDNA sequence of the CCS gene from the ATG initiation codon to a position 66 bp upstream has been described by Bouvier et al (The Plant Journal 6 (1) 45-54). The invention does not extend to these DNA sequences per se but does cover their use in the constructs, expression cassettes and the methods of the invention as described further herein.
xe2x80x9cActive variantsxe2x80x9d are DNA sequences partially homologous to SEQ ID NO 1 or SEQ ID NO 2 which retain promoter activity. It may be possible to alter the level or type of activity of these promoters by manipulating their sequences: for example, by altering the nucleotide sequence in key regulatory regions, by truncating the sequence or by deleting parts within the sequence.
The promoters of the invention are suitable for incorporation into DNA constructs encoding any target gene or transcribable DNA region so that the target gene is expressed when the construct is transformed into a plant. The DNA construct preferably contains a transcription termination signal.
The bell pepper FIB and CCS promoters may be synthesised ab initio using the sequence shown in SEQ ID NO 1 and SEQ ID NO 2 as a guide. Alternatively, the promoters may be isolated from plant genomic DNA libraries using suitable probes derived from the said sequences or the promoter may be isolated using a PCR approach.
In practice the promoter of the invention may be inserted as a promoter sequence in a recombinant gene construct designed for use in a plant. The construct is then inserted into the plant by transformation. Any plant species may be transformed with the construct, and any suitable transformation method may be employed. It is preferred that plants to be transformed with the promoters according to the present invention are plants containing chromoplasts.
According to a second aspect of the invention, there is provided a plant gene expression assette comprising the bell pepper FIB or CCS promoter operatively linked to a target gene, the promoter having the sequence shown as SEQ ID No 1, SEQ ID No 2 or active variants hereof.
The target gene is a DNA sequence which may be derived from an endogenous plant gene or from a foreign gene of plant, fungal, algal, bacterial, viral or animal origin. Normally it is a sequence other than the sequence encoding the FIB or CCS protein which follows the FIB or CCS promoter in the naturally occuring bell pepper FIB or CCS gene. The target gene may be a single gene or a series of genes. The target gene is adapted to be transcribed into functional RNA under the action of plant cell enzymes such as RNA polymerase. Functional RNA is RNA which affects the biochemistry of the cell: for example, it may be mRNA which is translated into protein by ribosomes or it may be RNA which inhibits the translation of mRNA related to it. Thus the target gene sequence may be a sense sequence encoding at least part of a functional protein or an antisense sequence.
The expression cassette is suitable for general use in plants. In practice the DNA construct comprising the expression cassette of the invention is inserted into a plant by transformation. Any transformation method suitable for the target plant or plant cells may be employed, including infection by Agrobacterium tumefaciens containing recombinant Ti plasmids, electroporation, microinjection of cells and protoplasts, microprojectile transformation, pollen tube transformation and transformation of plant cells using mineral fibres (U.S. Pat. No. 5,302,523, International Patent Application Publication Number W094/28148). The transformed cells may then in suitable cases be regenerated into whole plants in which the new nuclear material is stably incorporated into the genome. Both transformed monocotyledonous and dicotyledonous plants may be obtained in this way. Transgenic plant technology is for example described in the following publications: Swain WF, 1991, TIBTECH 9: 107-109; Ma JKC et al, 1994, Eur J Immunology 24: 131-138; Hiatt A et al, 1992, FEBS Letters 307:71-75; Hein MB et al 1991, Biotechnology Progress 7: 455-461; Duering K, 1990, Plant Molecular Biology 15: 281-294.
Examples of genetically modified plants which may be produced include but are not limited to field crops, cereals, fruit and vegetables such as: canola, sunflower, tobacco, sugarbeet, cotton, soya, maize, wheat, barley, rice, sorghum, mangoes, peaches, apples, pears, strawberries, bananas, melons, tomatoes, potatoes and carrot.
The invention further provides a plant cell containing a gene expression cassette according to the invention. The gene expression cassette may be stably incorporated in the plant""s genome by transformation. The invention also provides a plant tissue or a plant comprising such cells, and plants or seeds derived therefrom.
The invention further provides a method for controlling plant gene expression comprising transforming a plant cell with a plant gene expression cassette having a bell pepper FIB or CCS promoter operatively linked to a target gene, whereby the activated promoter drives expression of the target gene. The promoter may be activated under certain spatial, temporal, developmental and/or environmental conditions.
In order to determine their temporal and spatial expression, the promoter fragments of the bell pepper FIB and CCS genes are fussed to the GUS (xcex2-glucuronidase) reporter gene in DNA constructs suitable for plant transformation. GUS accumulation in transgenic plants may then be monitored.
xcex2-glucuronidase is a bacterial enzyme which catalyses the conversion of 5-Methylumbelliferyl glucuronide (MUG) to 4-Methylumbelliferone (MU) and Glucuronic acid. The conversion is measured by way of a fluorometer at 365 nm excitation 455 emission. A time course of the reaction can be carried out allowing the conversion rate to be measured in nanoMoles MU formed/mg protein/minute. This activity allows the analysis of gene expression controlled by the promoter in the transformed plants.